Prewetting lateral flow test strip

ABSTRACT

A test strip and method for detecting an analyte present in a sample. The test strip comprising: a buffer addition zone to which a buffer may be added; an absorbent zone proximal to the buffer addition zone; one or more test zones distal to the buffer addition zone, at least one of the test zones including a first analyte binding agent immobilized therein which is capable of binding to the analyte to be detected; a terminal buffer flow zone distal to the one or more test zones, the absorbent zone being positioned relative to the buffer addition zone and having an absorption capacity relative to the other zones of the test strip such that when a volume of buffer within a predetermined buffer volume range for the test strip is added to the buffer addition zone, a distal diffusion front of the buffer diffuses from the buffer addition zone to a distal diffusion point within the terminal buffer flow zone and then diffuses proximal relative to the one or more test zones; and a sample addition zone distal to the terminal buffer flow zone to which a sample may be added.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to lateral flow test strips and methods ofoperation for the lateral flow test strips.

2. Description of Related Art

Quantitative analysis of cells and analytes in fluid samples,particularly bodily fluid samples, often provides critical diagnosticand treatment information for physicians and patients. For example,immunological testing methods which take advantage of the highspecificity of antigen-antibody reactions, provide one approach tomeasurement of analytes. Kennedy, D. M. and S. J. Challacombe, eds.,ELISA and Other Solid Phase Immunoassays: Theoretical and PracticalAspects, John Wiley and Sons, Chichester (1988). This document and allothers cited to herein, are incorporated by reference as if reproducedfully below. Such assays may also find use in various otherapplications, such as veterinary, food testing, or agriculturalapplications.

Immunoassays that provide a quantitative measurement of the amount of ananalyte in a sample have previously used complex, multi-step proceduresand expensive analyzers available only in a laboratory setting.

Immunochromatographic assays, such as those described in GB 2,204,398A;U.S. Pat. Nos. 5,096,837, 5,238,652, and 5,266,497; Birnbaum, S. et al.,Analytical Biochem. 206:168-171 (1992); Roberts, M. A. and R. A. Durst,Analytical Chem. 67:482-491 (1995); and Klimov, A. D. et al., ClinicalChem. 41:1360 (1995), are simpler, yet do not provide a quantitativemeasurement of an analyte. Instead, these immunochromatographic assaysdetect the presence (or absence) of an analyte above a defined cutofflevel for the test performed. The lack of a quantitative measurementlimits the usefulness of these assays.

A variety of disposable diagnostic assay devices have also beendeveloped. Examples of such devices include, but are not limited toCathey, et al, U.S. Pat. No. 5,660,993; International Publication NumberWO 92/12428; Eisinger, et al, U.S. Pat. No. 4,943,522; Campbell, et al,U.S. Pat. No. 4,703,017; Campbell, et al, U.S. Pat. No. 4,743,560; andBrooks, U.S. Pat. No. 5,753,517. Nevertheless, a need still exists forimproved disposable diagnostic assay devices and methods.

SUMMARY OF THE INVENTION

Test strips are provided which are adapted to receive a buffer thatprewets the test strip and receive a sample which flows within theprewet test strip. The test strips are employed to detect one or moreanalytes that may be present in a sample.

According to one embodiment, the test strip comprises a buffer additionzone to which a buffer is added to prewet the test strip; an absorbentzone proximal to the buffer addition zone; one or more test zones distalto the buffer addition zone, at least one of the test zones including afirst analyte binding agent immobilized therein which is capable ofbinding to the analyte to be detected; and a terminal buffer flow zonedistal to the one or more test zones, the absorbent zone beingpositioned relative to the buffer addition zone and having an absorptioncapacity relative to the other zones of the test strip such that when avolume of buffer within a predetermined buffer volume range for the teststrip is added to the buffer addition zone, a distal diffusion front ofthe buffer diffuses from the buffer addition zone to a distal diffusionpoint within the terminal buffer flow zone and then diffuses proximalrelative to the one or more test zones. The test strip further comprisesa sample addition zone that is distal to the terminal buffer flow zone.When a sample is added to the sample addition zone, the sample diffuseswithin the test strip in a proximal direction across the terminal bufferflow zone, across the one or more test zones, and ultimately to theabsorbent zone. When the sample traverses the test zones, analyte in thesample is immobilized in whichever test zone(s) include(s) the firstanalyte binding agent bound therein.

The above described test strip may be used to detect an analyte in asample by a direct detection assay or may be used to detect an analytein a sample by a competitive assay. When the assay is a direct detectionassay, the amount of analyte in the sample is measured based on theamount of analyte which is immobilized in a test zone by a first analytebinding agent bound therein. When the assay is a competitive assay, thetest strip further comprises a competitive agent which is capable ofcompeting with the analyte for binding to the first analyte bindingagent. In this instance, the amount of analyte in the sample is measuredbased on how much less competitive agent is immobilized in the test zoneby the first analyte binding agent as compared to when a control isemployed as the sample which contains no analyte.

Control over the above described flow of the buffer within the teststrip (i.e., such that the buffer reaches the terminal buffer flow zoneand reverses the direction of buffer flow within the terminal bufferflow zone back toward the buffer addition zone and the absorbent zone)is achieved by controlling the amount of buffer added to the test stripwithin a predetermined range designed to be used with that test strip.

By adding the sample to the sample addition zone such that the samplereaches the terminal buffer flow zone after the buffer has reached theterminal buffer flow zone and has already reversed direction and isdiffusing back toward the absorbent zone, the sample is able to flowwithin a prewet test strip, thereby yielding more accurate and preciseresults.

As will be described in greater detail herein, depending on the layoutof the test strip, the buffer may be added before, at the same time, orafter the sample is added to the test strip. For example, the sampleaddition zone may be positioned relative to the test zones such thatsample is added to the sample addition zone at the same time that bufferis added to the buffer addition zone. The sample addition zone may alsobe positioned relative to the test zones such that sample added to thesample addition zone at the same time that the buffer is added to thebuffer addition zone. The sample addition zone may also be positionedrelative to the test zones such that the sample can be added to the teststrip before the buffer is added and nevertheless, the sample stillreaches the distal diffusion point of the buffer after the distaldiffusion front of the buffer has diffused to the distal diffusion zone,reversed direction and begun diffusing in a proximal direction.

According to any of the above test strip embodiments, 1, 2, 3 or moretest zones may be control zones with one or more control binding agentsimmobilized therein. The control zones may be used to calibrate the teststrip, may be used to confirm whether or not the test strip performed asintended, may be used detect whether too little or too much buffer wasadded and may be used to detect whether too little sample was added.

In one embodiment, the test strip comprises at least a first controlzone with a control binding agent immobilized therein. Optionally, thetest zones further includes a second control zone with a same controlbinding agent immobilized therein as the first control zone. The firstcontrol zone may contain the same or a different amount of the controlbinding agent than the second control zone. In a preferred embodiment,the first control zone contains about the same amount of the controlbinding agent as the second control zone.

Also according to any of the above test strip embodiments, a secondanalyte binding agent which is capable of binding to the analyte anddiffusing to the one or more test zones may be included on the teststrip. The second analyte binding agent is preferably incorporated onthe test strip adjacent either the sample addition zone or the bufferaddition zone, more preferably proximal relative to the sample additionzone or distal relative to the buffer addition zone such that additionof the sample or buffer causes the second analyte binding agent to becarried with the sample or buffer to the test zones.

The second analyte binding agent may also be delivered to the test stripvia the buffer or the sample, most preferably the sample. The secondanalyte binding agent may bind to components in the sample in additionto the analyte. Alternatively, the second analyte binding agent may bean agent which does not bind to components in the sample other than theanalyte.

In order to facilitate detection, the second analyte binding agent ispreferably labeled with a detectable marker. As discussed herein, any ofa wide range of detectable markers known in the art may be used. In apreferred embodiment, the second analyte binding agent is attached to aparticle which is capable of diffusing to the one or more test zones.The particle may serve as the detectable marker or may itself be labeledwith a detectable marker.

Also according to any of the above test strip embodiments, the teststrip may be for a competitive assay, in which case, the test strip mayinclude a competitive agent. The competitive agent may compete with theanalyte for binding to the first analyte binding agent.

The competitive agent is preferably incorporated on the test stripadjacent the sample addition zone, more preferably proximal relative tothe sample addition zone such that addition of the sample causes thecompetitive agent to be carried with the sample to the test zone.

Methods are also provided for detecting an analyte in a sample.

In one embodiment, the method comprises delivering a buffer to a teststrip which causes a distal diffusion front of the buffer to (a) diffusein a distal direction to one or more test zones, at least one of thetest zones including a first analyte binding agent immobilized thereinwhich binds to analyte in the sample, (b) diffuse to a terminal bufferflow zone distal to the one or more test zones, change direction and (c)diffuse to a position proximal to the one or more test zones; deliveringa sample to the test strip at a position distal to the terminal bufferflow zone, delivery of the sample causing analyte in the sample todiffuse proximally past the terminal buffer flow zone to the one or moretest zones after the distal diffusion front of the buffer diffusesproximal to the one or more test zones, the analyte binding to the firstanalyte binding agent and becoming immobilized in the test zones; anddetecting the analyte immobilized in the test zones.

According to the method, a second analyte binding agent may be presentwhich binds to the analyte. The second analyte binding agent may be usedto detect the immobilized analyte. The second analyte binding agent maybe contained on the test strip where the sample is delivered, deliveryof the sample causing the diffusion of the second analyte binding agent.Alternatively, the second analyte binding agent may be contained on thetest strip proximal to where the sample is delivered, delivery of thesample causing the diffusion of the second analyte binding agent.Delivering the sample to the test strip may also include delivering thesecond analyte binding agent to the test strip within the sample.

In another embodiment, the method is for a competitive assay. Accordingto this method, a buffer is delivered to a test strip which causes adistal diffusion front of the buffer to (a) diffuse in a distaldirection to one or more test zones, at least one of the test zonesincluding a first analyte binding agent immobilized therein which bindsto analyte in the sample, (b) diffuse to a terminal buffer flow zonedistal to the one or more test zones, change direction and (c) diffuseto a position proximal to the one or more test zones. A sample is alsodelivered to the test strip at a position distal to the terminal bufferflow zone such that delivery of the sample causes the sample diffuseproximally past the terminal buffer flow zone to the one or more testzones after the distal diffusion front of the buffer diffuses proximalto the one or more test zones.

Delivery of a sample to the test strip also causes a competitive agentto diffuse with the sample to the test zone. The competitive agentcompetes with the analyte for binding to the first analyte bindingagent. The competitive agent is preferably incorporated on the teststrip adjacent the sample addition zone, more preferably proximalrelative to the sample addition zone such that addition of the samplecauses the competitive agent to be carried with the sample to the testzone.

The method further comprises detecting the competitive agent immobilizedin the test zones. In order to facilitate detection, the competitiveagent is preferably labeled with a detectable marker.

According to any of the method embodiments, the buffer may be added tothe test strip at a same time as the sample is added to the test strip,before the sample is added to the test strip, or after the sample isadded to the test strip. When the sample is added to the test striprelative to the conjugate buffer depends on the time required for thebuffer to reach the terminal buffer flow zone which, in turn, depends onthe flow design of the test strip.

According to any of the above methods, the test zones may include afirst control zone with a control binding agent immobilized therein,delivering the buffer causing a control agent to diffuse distally to thefirst control zone and bind to the control binding agent immobilizedtherein. Alternatively, the test zones may include first and secondcontrol zones which each include an approximately the same orsignificantly different amount of a control binding agent immobilizedtherein, delivering the buffer causing a control agent to diffusedistally to the first and second control zones and bind to the controlbinding agent immobilized therein.

When one or more control zones are employed, a control agent may becontained on the test strip where the buffer is delivered, delivery ofthe buffer causing the diffusion of the control agent. Alternatively, acontrol agent may be contained on the test strip distal to where thebuffer is delivered, delivery of the buffer causing the diffusion of thecontrol agent. Delivering the buffer to the test strip may also includedelivering the control agent to the test strip—within the buffer.Incorporating the control agent into the buffer is advantageous becausevariability in the movement of control agents strip to strip arisingfrom differences in the way in which the control agents becomesresolubilized when buffer is added is avoided.

Also according to the above methods, detecting the second analytebinding agent may be facilitated by labeling the second analyte bindingagent with a detectable marker, detecting the second analyte bindingagent including detecting the detectable marker. The second analytebinding agent may be attached to a particle. Detecting the secondanalyte binding agent may include detecting the particle.

According to any of the above embodiments, the buffer delivered to thetest strip is preferably within a predetermined volume range that thetest strip has been designed to process. The predetermined volume rangeis preferably between about 10 and 250 L, preferably between about 20and 200 L, more preferably between about 20 and 100 L, and mostpreferably between about 40 and 60 L. When a buffer is delivered to thetest strip within the predetermined volume range, the terminal sampleflow zone may be designed to have a short length from a proximal end toa distal end. For example, when a buffer is delivered to the test stripwithin a range of about 35 and 45 L, the terminal flow zone may have alength from a proximal end to a distal end of between about 1 and 25 mm,more preferably 2 and 15 mm, and most preferably 3 and 10 mm.

Also according to any of the above embodiments, the first analytebinding agent preferably does not bind to components in the sample otherthan the analyte. Types of molecules that can serve as first analytebinding agents include, but are not limited to antibodies, engineeredproteins, peptides, haptens, lysates containing heterogeneous mixturesof antigens having analyte binding sites, ligands and receptors. In oneparticular embodiment, the first analyte binding agent is an antibody orfragment thereof.

Also according to any of the above embodiments, the buffer added to thebuffer addition zone may comprise the sample being tested. Optionally,the buffer may be the sample. When sample forms all or a portion of thebuffer that is added to buffer addition zone, the buffer still performsthe function of prewetting the test strip. The ability to use sample, inwhole or in part, as the buffer allows the present invention to moreeasily accommodate a wider range of sample and external liquid controlmatrices (e.g., serum, plasma, euglobulin). In addition, differences inflow behavior within the test strip between sample and buffer can bereduced by adding the same composition (e.g., the sample) to both thesample and buffer addition zones.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a top-down view of an embodiment of a lateral flowtest strip according to the present invention.

FIGS. 2A-2H illustrate a method of operation for a lateral flow teststrip according to the present invention.

FIG. 2A illustrates a buffer being added to the test strip.

FIG. 2B illustrates the buffer flowing within the test strip.

FIG. 2C illustrates the test strip when the buffer has flowed a distancewithin the test strip in the direction opposite an absorbent zone towithin a terminal buffer flow zone.

FIG. 2D illustrates the test strip where the buffer is flowing backtoward the absorbent zone.

FIG. 2E illustrates the addition of a sample to the test strip.

FIG. 2F illustrates the flow of the sample within the test strip towardthe absorbent zone.

FIG. 2G illustrates the flow of the sample within the test strip pastthe test zone.

FIG. 2H illustrates the flow of the sample within the test strip intothe absorbent zone.

FIGS. 3A-3H a method of operation for a lateral flow test stripaccording to the present invention.

FIG. 3A illustrates a sample and buffer being added to the test strip.

FIG. 3B illustrates the sample and buffer flowing within the test strip.

FIG. 3C illustrates the test strip when the buffer has flowed a distancewithin the test strip in the direction opposite an absorbent zone to ato within terminal buffer flow zone.

FIG. 3D illustrates the test strip where the buffer is flowing backtoward the absorbent zone.

FIG. 3E illustrates the sample continuing to flow toward the bufferflow.

FIG. 3F illustrates the sample having flowed past the terminal bufferflow zone.

FIG. 3G illustrates the flow of the sample within the test strip pastthe test zone.

FIG. 3H illustrates the flow of the sample within the test strip intothe absorbent zone.

FIG. 4 illustrates a test strip design where the sample addition zone ispositioned adjacent the buffer addition zone.

FIGS. 5A-5C illustrate various cartridge designs into which a test stripaccording to the present invention can be positioned.

FIG. 5A illustrates a cartridge design adapted for the test stripillustrated in FIGS. 2A-2H.

FIG. 5B illustrates a cartridge design adapted for the test stripillustrated in FIGS. 3A-3H where the buffer addition zone is positionedan extended distance from the sample addition zone such that the sampleand wash buffer can be added at the same time.

FIG. 5C illustrates a cartridge design adapted for the test stripillustrated in FIG. 4 where the sample addition zone is positionedadjacent the buffer addition zone, the test zone being positioned anextended distance from the sample addition zone.

FIG. 6A illustrates the layout of a FLEXPACKJHP test strip manufacturedby Abbott.

FIG. 6B illustrates the operation of the test strip illustrated in FIG.6A.

FIG. 7 illustrates a side break-away view of the lateral flow test stripillustrated in FIG. 1.

FIG. 8 illustrates the results from the TSH assay performed in Example2.

FIG. 9 shows a standard curve derived from the TSH assay results shownin FIG. 8.

FIG. 10 illustrates the results from the PSA assay performed in Example3.

FIG. 11 shows a standard curve derived from the PSA assay results shownin FIG. 10.

FIG. 12 illustrates a comparison between the performance of the ReLIA™TSH assay when sample is added to both the top and bottom ports and whensample is added only to the bottom port.

FIG. 13 illustrates the reproducible of measuring TSH levels using thetest strip of example 4.

FIG. 14 illustrate TSH values relative to a standard curve for the teststrip of example 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to lateral flow test strips and methodsfor employing such test strips which exhibit greater precision andaccuracy. More specifically, the lateral flow test strips and methods ofthe present invention reduce performance variability, most likely due tointerferences that might affect the absolute amount of binding of eitheranalyte binding agent or control binding agent to a test zone, caused byvariations in liquid flow rates across the test strip.

The present invention addresses the problem that the flow rate of a wettest strip is significantly different than the flow rate of a dry teststrip. For example, fluid tends to flow faster when the test strip isdry than when it is wet. In order to minimize these flow rateinfluences, the present invention provides test strips which aredesigned to be prewet prior to the addition of a sample, therebyequilibrating the flow rate of the test strip so that a sample, onceadded, moves through the test strip at a more uniform rate across thetest strip. In one embodiment, the test strip is prewet using the samesample that is being tested. By using the same sample as both aprewetting solution and as a sample, flow rate differences are furtherminimized.

Given that test strips need to yield reliable and consistent resultsindependent of the person using the test strips, an important aspect ofthe present invention is the simplicity with which a test strip may beprewet to afford more uniform sample velocity. As will be discussedherein in greater detail, the design of the test strips of the presentinvention cause a prewetting solution, referred to herein as a buffer,to flow across the one or more test zones and control zones and thenindependently flow back toward the buffer addition zone withoutunintended portions of the strip becoming wet. This controlled flow andprewetting of the test strip accomplishes the desired results ofproviding a test strip that may be used with consistency,reproducibility and eliminates the need for operator intervention.

A further feature of the test strips of the present invention is thereduced timing sensitivity of the test strips regarding when buffer andsample is added to the test strip. Instead, the test strips of thepresent invention allow sample to be added within a broader time windowafter buffer is added.

Other factors influencing lateral flow test results include: 1)variability in the release of an analyte binding agent or the controlagent from a conjugate pad, 2) device to device variation in thenon-specific binding of the analyte binding population to the teststrip, 3) variability in the movement of the analyte binding populationthrough or along the test strip during the assay due to variation in thepore size of the test strip or membrane strip materials or non-specificaggregation of the analyte binding agent. These other sources ofvariability are also reduced by the test strips of the presentinvention.

According to one embodiment, a test strip is provided which comprises abuffer addition zone to which a buffer is added to prewet the teststrip; an absorbent zone proximal to the buffer addition zone; one ormore test zones distal to the buffer addition zone, at least one of thetest zones including a first analyte binding agent immobilized thereinwhich is capable of binding to the analyte to be detected; and aterminal buffer flow zone distal to the one or more test zones. Theabsorbent zone is positioned relative to the buffer addition zone andhas an absorption capacity relative to the other zones of the test stripsuch that a distal diffusion front of a buffer added to the bufferaddition zone diffuses from the buffer addition zone to a distaldiffusion point within the terminal buffer flow zone and then reversesdirection, independent of any user intervention, and diffuses proximalrelative to the one or more test zones.

The independent flowing back of the buffer toward the buffer additionzone is achieved by positioning an absorbent zone relative to the bufferaddition zone such that when a volume of buffer (within a predeterminedvolume range for that test strip) is added to the test strip, thediffusion front of the buffer expands across the one or more test zonesto a terminal buffer flow zone. When the buffer reaches the terminalbuffer flow zone, the absorbent properties of the absorbent zone causesthe buffer to be drawn backward across the test zones toward the bufferaddition zone and ultimately into the absorbent zone.

By causing buffer to flow across the one or more test zones and thenindependently flow back toward the buffer addition zone, the test stripis effectively prewet prior to the addition of sample. As a result, whensample is added to the test strip, the sample is believed to flow withinthe test strip at a more consistent velocity, thereby yielding moreconsistent results.

The ability to cause the buffer to flow back toward the buffer additionzone independent of any user interaction reduces the time criticality ofwhen sample is added to the test strip. As will be discussed herein ingreater detail, the self-timing features of test strips according to thepresent invention provides several significant advantages over previoustest strips.

The test strip also comprises a sample addition zone that is distal tothe terminal buffer flow zone. When a sample is added to the sampleaddition zone, the sample diffuses within the test strip in a proximaldirection across the terminal buffer flow zone, across the one or moretest zones, and ultimately to the absorbent zone. Analyte in the samplebinds to one or more test zones and is detected there.

FIG. 1 illustrates a top-down view of an embodiment of a lateral flowtest strip 100 according to the present invention. As illustrated, thetest strip 100 has proximal and distal ends 102, 104 respectively andcan be divided into several different zones. The test strip includes abuffer addition zone 106 where a buffer may be added to the test strip100. An absorbent zone 108 is positioned proximal to the buffer additionzone 106. One or more test zones 110, 112, 114 are positioned distal tothe buffer addition zone 106. The test strip 100 also includes aterminal buffer flow zone 116 distal to the one or more test zones 110,112, 114. Each of the above mentioned zones are in fluid diffusioncommunication with each other.

As illustrated, the test strip also includes a sample addition zone 118distal to the terminal buffer flow zone 116. The sample addition zone118 may be a zone where sample may be added to the test strip.Alternatively, the sample addition zone 118 may simply correspond to azone to which sample diffuses from a more distal point on the teststrip.

The test strip may also include a zone distal to the terminal bufferflow zone 116 which includes either a second analyte binding agent inthe case of a direct assay or a competitive agent in the case of acompetitive assay. In FIG. 1, the sample addition zone 118 may serve asthe zone comprising the second analyte binding agent or the competitiveagent. Alternatively, the zone comprising the second analyte bindingagent or the competitive agent may be proximal to the sample additionzone 118.

It is noted that the layout of the test strip illustrated in FIG. 1 islinear in design. However, non-linear layouts, such as the layoutillustrated in FIG. 4, are also intended for the test strips accordingto the present invention.

FIGS. 2A-2H illustrate a method of operation of a lateral flow teststrip, such as the one illustrated in FIG. 1. Prior to performing anassay using a test strip according to the present invention, a fluidsample is obtained that is believed to contain the analyte to bedetected. The sample can include any fluid that wets the test strip andhas a viscosity that is sufficient to allow movement of the sampleacross the test strip. In a preferred embodiment, the sample is anaqueous solution (such as a bodily fluid).

Also prior to performing an assay, buffer is obtained which is to beadded to the test strip. As described herein, the buffer may optionallycontain a control agent. Incorporating the control agent into the bufferis advantageous because variability in the movement of control agentsstrip to strip arising from differences in the way in which the controlagents becomes resolubilized when buffer is added is avoided. As alsodescribed herein, the buffer added to the buffer addition zone maycomprise the sample being tested. Optionally, the buffer may be thesample. When sample forms all or a portion of the buffer that is addedto buffer addition zone, the buffer still performs the function ofprewetting the test strip. The ability to use sample, in whole or inpart, as the buffer allows the present invention to more easilyaccommodate a wider range of sample and external liquid control matrices(serum, plasma, euglobulin). In addition, differences in flow behaviorwithin the test strip between sample and buffer can be reduced by addingthe same composition (e.g., the sample) to both the sample and bufferaddition zones.

FIG. 2A illustrates buffer 120 being added to the buffer addition zone106 of the test strip 100. It is noted that the test strip is designedfor use with a volume of buffer that is within a particular volumerange. More specifically, delivering buffer to the buffer addition zonewithin the predetermined volume range causes the buffer to diffusedistally beyond the test zones into the terminal buffer flow zone 116,but not beyond the terminal buffer flow zone 116 (as illustrated in FIG.2D).

As illustrated in FIG. 2B, the buffer 120 begins to diffuse bothproximally and distally across the test strip after being added to thetest strip. As illustrated in FIG. 2C, the distal front 124 of thebuffer 120 diffuses across the one or more test zones 110, 112, 114 towithin the terminal buffer flow zone 116. As illustrated in FIG. 2D, thedistal front 124 of the buffer 120 ultimately extends to a point withinthe terminal buffer flow zone 116.

When the volume of the buffer added to the test strip is within apredetermined volume range for which the test strip is designed, thedistal front 124 of the buffer 120 reaches a distal diffusion pointcorresponding to a point of maximum distal flow somewhere within theterminal buffer flow zone 116. At this point, as illustrated in FIG. 2E,capillary action by the absorbent zone 108 draws the buffer proximallytoward the absorbent zone 108. As the buffer is drawn into the absorbentzone 108, the distal front 124 of the buffer recedes proximally.

As can be seen from FIGS. 2A-2D, a feature of the present invention isthe control of where and how the buffer flows within the test strip. Thebuffer delivered to the test strip is preferably within a predeterminedvolume range that the test strip has been designed to process. Thepredetermined volume range is preferably between about 10 and 250 L,preferably between about 20 and 200 L, more preferably between about 20and 100 L, and most preferably between about 40 and 60 L. When buffer isdelivered to a test strip within these ranges, the flow of the bufferstops within the terminal buffer flow zone.

The terminal buffer flow zone may be designed to have a short lengthfrom a proximal end to a distal end. For example, when buffer isdelivered to the test strip within a range of about 35 and 45 L, theterminal buffer flow zone may have a length from a proximal end to adistal end of between about 1 and 25 mm, more preferably 2 and 15 mm,and most preferably 3 and 10 mm.

Positioned within one of the test zones (e.g., test zone 112) is a firstanalyte binding agent which binds to an analyte in a sample which thetest strip is designed to detect. Analyte present in the portion of thesample which flows across the test zones is immobilized in test zone 112by the first analyte binding agent.

FIG. 2E illustrates the addition of a sample 122 to the test strip atthe sample addition zone 118 after the buffer has reached the terminalbuffer flow zone. The volume of sample added is preferably between about10 and 250 L, preferably between about 20 and 150 L, more preferablybetween about 50 and 150 L, and most preferably between about 75 and 125L. It is noted that the most preferred volume of sample to add to a teststrip will vary depending on the assay.

The sample 122 may contain one or more different second analyte bindingagents which can bind to the analyte and enable analyte immobilized inthe test zones to be detected. It is noted that the sample addition zone118 may optionally include the one or more second analyte binding agentsused to detect immobilized analyte. In that instance, addition of thesample 122 serves to initiate diffusion of the one or more secondanalyte binding agents across the test zones.

As illustrated in FIGS. 2F and 2G, the sample 122 flows proximallyacross the test strip toward the absorbent zone 108, thereby causingboth analytes in the sample and the one or more second analyte bindingagents to move across the test zones 110, 112, 114 and bind toimmobilized analyte.

As illustrated in FIG. 2H, capillary action by the absorbent zone 108causes the buffer 120 to diffuse into the absorbent zone 108. Meanwhile,the sample 122 continues to diffuse proximally across the test zones110, 112, 114 and into the absorbent zone 108. Any of the one or moresecond analyte binding agents that were not immobilized in the testzones 110, 112, 114 are carried with the sample 122 into the absorbentzone 108.

In regard to the embodiment illustrated in FIGS. 2A-2H, it is noted thatthe sample 122 should be added to the test strip after the buffer 120has reached the test zones 110, 112, 114 and preferably after the bufferhas reached the terminal buffer flow zone 116 and has begun to diffuseback toward the absorbent zone 108. This allows the buffer 120 to prewetthe test strip.

FIGS. 3A-3H illustrate an alternative test strip design and method ofoperation for the test strip. In this embodiment, the buffer and sampleare added at the same time. In order for the buffer and sample to beadded at about the same time, it is necessary for the sample to reachthe test zones 210, 212, 214 after the buffer has contacted the testzones. It is preferred that the sample reach the test zones after thebuffer has begun diffusing back across the test zones toward theabsorbent zone 208.

Delaying when the sample reaches the test zones is accomplished in thisembodiment by creating a longer distance between sample addition zone218 and the terminal buffer flow zone 216 as compared to the test stripdesign illustrated in FIGS. 2A-2H. Alternatively, one can use a materialwhich causes the sample to diffuse at a slower rate.

FIG. 3A illustrates a buffer 220 being added to a buffer addition zone206 of the test strip 200. Meanwhile, a sample 222 is added to a sampleaddition zone 218 at about the same time that the buffer is added to thetest strip.

As illustrated in FIG. 3B, the buffer 220 begins to diffuse bothproximally and distally within the test strip once added to the teststrip. Meanwhile, the sample 222 also diffuses proximally and optionallydistally within the test strip.

As illustrated in FIG. 3C, the distal front 224 of the buffer 220diffuses across one or more test zones 210, 212, 214 to within aterminal buffer flow zone 216. Meanwhile, the sample 222 continues todiffuse proximally within the test strip toward the test zones.

As illustrated in FIG. 3D, the distal front 224 of the buffer 220ultimately extends to a point within the terminal buffer flow zone 216.At the time when the buffer is in the terminal buffer flow zone 216, thesample 222 has not yet reached that zone.

As illustrated in FIG. 3E, capillary action by the absorbent zone 208draws the buffer proximally toward the absorbent zone 208. As the bufferis drawn into the absorbent zone 208, the distal front 224 of the bufferflows proximally.

FIG. 3F illustrates the sample 222 reaching the test zones. As can beseen, by the time the sample 222 reaches the test zones, the distalfront 224 of the buffer has already flowed proximally out of theterminal sample flow zone 216 and the test zones 210, 212, 214.Positioned within one of the test zones (e.g., test zone 212) is a firstanalyte binding agent which binds to analyte in the sample which thetest strip is designed to detect. Analyte present in the portion of thesample which flows across the test zones is immobilized in test zone 212by the first analyte binding agent.

As illustrated in FIGS. 3G and 3H, capillary action by the absorbentzone 208 causes the buffer to withdraw into the absorbent zone 208.Meanwhile, the sample 222 continues to diffuse proximally across thetest zones 210, 212, 214 and into the absorbent zone 208. Any of the oneor more second analyte binding agents that were not immobilized in thetest zones 210, 212, 214 are carried with the sample 222 into theabsorbent zone 208.

The sample 222 added to the test strip may contain one or more secondanalyte binding agents which can bind to the analyte and enable analyteimmobilized in the test zones to be detected. Alternatively, the teststrip may include a conjugate zone distal to the terminal buffer flowzone 216 which contains one or more second analyte binding agents. Thesample addition zone 218 may also serve as the conjugate zone. When theone or more second analyte binding agents are preloaded onto the teststrip, the sample 222 serves to initiate diffusion of the one or moresecond analyte binding agents across the test zones toward the absorbentzone.

As illustrated in FIGS. 3A-3H, the sample may be added to the test stripbefore the buffer reaches the test zones by designing the diffusion pathof the test strip such that the sample does not reach the test zonesuntil after the buffer has diffused over and then back from the testzones. It is noted that the diffusion of the sample to the test zonesmay be sufficiently delayed that one adds the sample to the test stripprior to adding the buffer to the test strip.

In regard to the embodiments illustrated in FIGS. 2A-2H and 3 A-3H, itis noted that the method is a direct assay, i.e., the amount of analytepresent is measured by measuring the amount of analyte immobilized in atest zone. Competitive assays, i.e., assays where the amount of analytepresent is measured by measuring how much less of a competitive agent isimmobilized in a test zone. In order to perform a competitive assay, theoperation of the test strips illustrated in FIGS. 2A-2H and 3A-3H needonly be modified by employing a competitive agent which competes withthe analyte to bind to the first analyte binding agent.

FIG. 4 illustrates an alternative test strip design for a lateral flowtest strip according to the present invention. The operation of the teststrip is similar to the operation described in FIGS. 3A-3H. The samereference numerals are employed in FIG. 4 as in FIGS. 3A-3H. Asillustrated in FIG. 4, the buffer addition zone 206 is positionedadjacent the sample addition zone 218. This allows for a more compacttest strip design while also allowing the sample and buffer to be addedsimultaneously.

One feature of the test strip design illustrated in FIG. 4 is that thesample and buffer are added to the same end of the test strip. It isalso noted that the test zones 210, 212, 214 are positioned toward anopposite end of the sample and buffer addition zones 206, 218. Thismakes it possible for the test zones to be positioned within a samplereader while the sample and buffer addition zones are outside the samplereader. This, in turn, allows sample and buffer to be added to the teststrip while the test strip is in a test strip reader.

FIGS. 5A-5C illustrate various cartridge designs into which test stripsaccording to the present invention can be positioned. In each cartridgedesign, the cartridge includes a buffer addition port 240 adjacent thebuffer addition zone 206 of the test strip. The cartridge also includesa sample addition port 242 adjacent the sample addition zone 218 of thetest strip. The cartridge also includes a test window 244 adjacent thetest zones 210, 212, 214 of the test strip.

FIG. 5A illustrates a cartridge design adapted for the test stripillustrated in FIGS. 2A-2H. FIG. 5B illustrates a cartridge designadapted for the test strip illustrated in FIGS. 3A-3H where the bufferaddition zone is positioned an extended distance from the sampleaddition zone such that the sample and buffer can be added to the teststrip at about the same time. FIG. 5C illustrates a cartridge designadapted for the test strip illustrated in FIG. 4 where the bufferaddition zone is positioned adjacent the sample addition zone, the testzone being positioned an extended distance from the sample additionzone.

It is noted with regard to FIGS. 2-4 that a feature of the test stripsof the present invention is the test strip's inherent ability to exposetest zones on the test strip to buffer for a period of time and then tocause the buffer to diffuse away from the test zones prior to the samplereaching the test zones. This feature is made possible by matching (1)the positioning of the absorbent zone relative to the buffer additionzone with (2) the absorbent capacity of the test strip between thebuffer addition zone and the terminal buffer flow zone and (3) thevolume of the buffer to be delivered to the test strip. If too muchbuffer is delivered, the buffer will diffuse beyond the terminal bufferflow zone. If too little buffer is delivered, the buffer does notdiffuse far enough in the test strip to reach the test zones and thusdoes not adequately prewet the test strip.

The test strip's ability to expose the test zones to buffer for alimited period of time and then cause the buffer to be removed from thetest zones confers a timing independence to the test strip whichenhances the test strip's precision and ease of use. For example, testresults are not dependent on when the sample and buffer are added to thetest strip. As a result, the test strips need not be carefully monitoredregarding when the sample should be added. In this regard, the window oftime after the buffer has been added when sample should be added to thetest strip is substantially eliminated by the present invention.

The dynamics of using the volume of the buffer delivered to the teststrip to control how the buffer diffuses within the test strip will nowbe illustrated in regard to FIG. 1. As discussed previously, FIG. 1illustrates a test strip which has proximal and distal ends 102, 104respectively and is divided into several distinct zones. The test stripincludes a buffer addition zone 106 where a buffer is added to the teststrip. An absorbent zone 108 is positioned proximal to the bufferaddition zone 106. A test zone 112 is positioned distal to the bufferaddition zone 106. A terminal buffer flow zone 116 is positioned distalto the test zone 112. A sample addition zone 118 is positioned distal tothe terminal buffer flow zone 116.

For the purpose of illustration, assume that the test zone 112 includesa first analyte binding agent and the sample addition zone 118 includesa second analyte binding agent labeled with a detectable marker. Alsoassume that the test strip is designed such that a buffer volume of 30 Lwill cause the buffer to diffuse to but not beyond the test zone 112.Meanwhile, a buffer volume of 50 L will cause the buffer to diffuse tothe distal end of the terminal buffer flow zone 116.

If buffer is delivered to the test strip within the 30-50 L volumerange, the distal front of the buffer will diffuse past the test zone112. Distal advancement of the buffer will stop within the terminalbuffer flow zone 116. The buffer then flows back in the proximaldirection toward the absorbent zone 108 past the test zone 112, therebyprewetting the test strip. When the sample is added, the sample causesthe analyte in the sample and the second analyte binding agent todiffuse across the test zone 112. The second analyte binding agent bindsto the analyte which in turn binds to the first analyte binding agentimmobilized in the test zone 112. Other components in the sample willnot bind to the first analyte binding agent antibody since the firstanalyte binding agent is selective for the analyte. Since the bufferdiffuses away from the test zone 112 prior to the sample reaching thetest zone 112, the prior addition of the buffer prewets the test stripbut the flow of the buffer does not interfere with the flow of thesample within the test strip.

If a buffer volume of less than 30 L is delivered (e.g., 25 L) to thetest strip, the buffer never diffuses to the test zone 112. As a result,the buffer does not prewet the test strip in the test zone 112. When thesample is added, the sample has to flow across a combination of dry teststrip and wet test strip which can create variations due to differencesin flow rates.

If the buffer volume delivered is greater than 50 L (e.g., 55 L), thebuffer will diffuse past the test zone 112 and past the terminal bufferflow zone 116 into the sample addition zone. When too much buffer isadded, the test strip could be flooded, thereby interfering with thetest strip's operation. Also, in some embodiments, the buffer couldcause diffusion of a second analyte binding agent or a competitive agentpositioned distal relative to the terminal buffer flow zone 116.

As has been described above, one of the advantages of the test strips ofthe present invention is their self-timing property. In order to explainthe significance of these properties, a comparison will now be made tothe FLEXPACKJHP test strip manufactured by Abbott which is illustratedin FIGS. 6A and 6B.

FIG. 6A illustrates the layout of the test strip. As illustrated, thetest strip includes two separate sections 310, 312 which are attached toeach other by a hinge 314. Section 310 on the right includes a teststrip 316 which includes a sample addition zone 318, a test zone 319, alimit line 320, and a conjugate buffer transfer pad 322. Section 312 onthe left includes an absorbent pad 324 which is positioned opposite thesample addition zone 318, a conjugate buffer addition pad 326 which ispositioned opposite the conjugate buffer transfer pad 322, and a testwindow 328 which is positioned opposite the test zone 319. The opposingpositionings of the absorbent pad 324, the conjugate buffer addition pad326, and the test window 328 allows the absorbent pad 324 to contact thesample addition zone 318 and the conjugate buffer addition pad 326 tocontact the conjugate buffer transfer pad 322 when the first and secondsections 310, 312 are brought into contact with each other. In addition,the test zone 319 can be seen through the test window 328 when the firstand second sections 310, 312 are brought together.

FIG. 6B illustrates the operation of the test strip illustrated in FIG.6A. As illustrated, a conjugate buffer 330 is added to the conjugatebuffer addition pad 326. The conjugate buffer addition pad 326 includesa second analyte binding agent (e.g., an antibody) capable of binding toan analyte in the sample to be detected. The second analyte bindingagent is labeled with a detectable marker which allows the secondanalyte binding agent to be visualized. The second analyte binding agentis not specific for the analyte and thus can bind to other components inthe sample.

A sample 332 is then taken and added to the sample addition zone 318.Once added, the sample diffuses through the test strip 316 from thesample addition zone 318 across the test zone 319. The test zone 319includes an immobilized first analyte binding agent (e.g., an antibody)which selectively binds to an analyte in the sample which the test stripis designed to detect. When the sample traverses the test zone 319,analyte in the sample binds to the first analyte binding agent and isimmobilized in the test zone 319.

When the diffusion front of the sample reaches the limit line 320, theuser is supposed to bring the first and second sections 310, 312together. Bringing the first and second sections 310, 312 togethercauses the absorbent pad 326 to draw the sample back toward the sampleaddition zone 318. Meanwhile, conjugate buffer is transferred to theconjugate buffer transfer pad 322 from the conjugate buffer addition pad320. The conjugate buffer diffuses from the conjugate buffer transferpad 322 across the test zone 319. Second analyte binding agent that wasstored in the conjugate buffer addition zone 318 diffuses with theconjugate buffer and contacts immobilized analyte in the test zone 319.Observation of the visually detectable marker on the second analyte,binding agent once immobilized in the test zone 319, is used to detectthe analyte.

As can be seen from the above description of the operation of theFLEXPACKJHP test strip, it is necessary to determine when the samplereaches the limit line 320 before causing the conjugate buffer to betransferred from the buffer addition zone 318 to the conjugate bufferaddition pad 320 and begin flowing toward the test zone 319. It is alsonecessary to take the affirmative step of contacting the sample additionzone 318 with the absorbent pad 324 in order to cause the sample to bewithdrawn from the test zone 319.

The design of the test strips of the present invention, for examplethose illustrated in FIGS. 2-4, eliminate the need to monitor the teststrip to determine when to begin the removal of the sample from the testzone. It is noted that no monitoring is required and that the sample isadded after buffer in the test strips of the present invention asopposed to the FLEXPACKJHP test strip.

In addition, since the buffer withdraws automatically, one need notcarefully monitor the test strip regarding when to add the sample.Rather, test results using the test strips of the present invention arenot dependent on when the sample reaches the test zones after the bufferdiffuses from the test zones.

Lateral flow assays according to the invention may find use in a varietyof applications. For example, the assays may be used to assay for humandiseases, such as infectious diseases, or any other human diseasesinvolving recognizable epitopes (e.g. cancer, autoimmune disease,cardiovascular conditions, hormone testing, and pathology). The assaysmay also be used in veterinary, food testing, agricultural, or finechemical applications. The lateral flow assays according to theinvention may be performed in variety of ways, including use of alateral flow assay testing apparatus, such as that disclosed in theapplication Ser. No. 09/199,255, filed Nov. 23, 1998 which isincorporated herein by reference. In a preferable embodiment, thelateral flow assay testing apparatus comprises a ReLIAJ testingapparatus, available from PraxSys BioSystems (San Ramon, Calif.).

1. Construction of Test Strips According to the Present Invention

Methods and materials for constructing test strips according to thepresent invention will now be discussed in greater detail. It is notedthat the particular construction of the test strip may be varied,depending on the particular assay that the test strip is intended toperform. Variations in the way in which the test strips may beconstructed beyond this example are intended to fall within the scope ofthe invention.

FIG. 7 illustrates a side break-away view of the lateral flow test stripillustrated in FIG. 1. As illustrated in FIG. 7, the test strip 100 mayinclude a backing strip 402 which runs a length of the test strip. Amembrane strip 404 is positioned over the backing strip 402 and servesas a diffusion passageway for the test strip. An absorbent pad 408 ispositioned over the membrane strip 404 within the absorbent zone 108which is positioned toward a proximal end of the test strip. A bufferpad 406 is positioned over the membrane strip 404 distal to theabsorbent pad 408. An adhesive 409 may be used to attach the buffer pad406 to the membrane strip 404. One or more test zones 410, 412, 414 maybe formed in the membrane strip 404 distal to the sample pad 406. Someof these test zones may be control zones and some may be for measuringan analyte in the sample. A conjugate pad 416 is positioned over themembrane strip 404 distal to the test zones 410, 412, 414 and distal tothe terminal buffer flow zone 116. A sample pad is positioned over ordistal to the conjugate pad. A protective cover 418 optionally may bepositioned over the test zones.

The backing strip may be made of any stable, non-porous material that issufficiently strong to support the materials and strips coupled to it.Since many assays employ water as a diffusion medium, the backing stripis preferably substantially impervious to water. In a preferredembodiment, the backing strip is made of a polymer film, more preferablya polyvinyl film.

The membrane strip may be made of any substance which has sufficientporosity to allow capillary action of fluid along its surface andthrough its interior. The membrane strip should have sufficient porosityto allow movement of antibody- or antigen-coated particles. The membranestrip should also be wettable by the fluid used in the sample whichcontains the analyte to be detected (e.g., hydrophilicity for aqueousfluids, hydrophobicity for organic solvents). Hydrophobicity of amembrane can be altered to render the membrane hydrophilic for use withaqueous fluid, by processes such as those described in U.S. Pat. No.4,340,482, or U.S. Pat. No. 4,618,533, which describe transformation ofa hydrophobic surface into a hydrophilic surface. Examples of substanceswhich can be used to form a membrane strip include: cellulose,nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyteion exchange membrane, acrylic copolymer/nylon, and polyethersulfone. Ina preferred embodiment, the membrane strip is made of nitrocellulose.

The absorbent pad may be formed of an absorbent substance that canabsorb the fluid used as the sample and buffer. The absorption capacityof the absorbent pad should be sufficiently large to absorb the fluidsthat are delivered to the test strip. Examples of substances suitablefor use in an absorbent pad include cellulose and glass fiber.

The sample and buffer addition pads may be formed of any absorbentsubstance. Examples of substances that may be used include cellulose,cellulose nitrate, cellulose acetate, glass fiber, nylon,polyelectrolyte ion exchange membrane, acrylic copolymer/nylon, andpolyethersulfone.

As discussed previously, the sample addition pad may serve as theadditional role of being the conjugate pad and contain an agent labeledwith a detectable marker which is capable of binding to the analyte tobe detected in the sample. In competitive assays, the sample additionpad may contain a competitive agent. Alternatively, the test strip mayinclude a conjugate pad separate from the sample addition pad whichcontains an agent labeled with a detectable marker which is capable ofbinding to the analyte to be detected in the sample. In competitiveassays, the conjugate pad may contain a competitive agent. In FIG. 7, aconjugate pad is shown as element 420 beneath the sample addition pad416. It is noted that the conjugate pad in FIG. 7 is positioned in theflow path between the sample addition pad 416 and the remainder of thetest strip.

The protective cover, if used, may be formed of any material which isimpervious to water, and is preferably translucent or transparent. Theprotective covering may be a single or multiple layers. Preferablematerials for use in the protective covering include opticallytransmissive materials such as polyamide, polyester, polyethylene,acrylic, glass, or similar materials. The protective covering may beclear or not clear depending on method of detection used. In apreferable embodiment, protective covering is optically clear polyester.

2. Assays for Use with Test Strips According to the Present Invention

The test strips of the present invention are intended to be employablewith a wide variety of lateral flow assays involving two analyte bindingagents which each can bind to an analyte to be detected. At least one ofthe binding agents should bind selectively to the analyte. Morespecifically, one of the binding agents should bind to the analyte andnot bind to any other components of the sample.

As used herein, the term, “analyte,” is intended to refer to anycomponent of a sample (e.g., molecule, compound, or aggregate thereof)which is to be detected and optionally quantitatively determined by anassay test strip. Examples of analytes include proteins, such ashormones and other secreted proteins, enzymes, and cell surfaceproteins; glycoproteins; peptides; small molecules; polysaccharides;antibodies (including monoclonal or polyclonal Ab and portions thereof);nucleic acids; drugs; toxins; viruses or virus particles; portions of acell wall; and other compounds possessing epitopes.

The first and second analyte binding agents may be any agents which canbind to the analyte to be detected. A variety of different types ofmolecules can be used as analyte binding agents, including, for example,antibodies, engineered proteins, peptides, haptens, and lysatescontaining heterogeneous mixtures of antigens having analyte bindingsites. P. Holliger et al., Trends in Biotechnology 13:7-9 (1995); S. M.Chamow et al., Trends in Biotechnology 14:52-60 (1996). If the analyteto be detected is a ligand, a receptor which binds to the ligand can beused, and vice versa. In one particular embodiment, the first and/orsecond analyte binding agents are antibodies which bind to animmunogenic portion of the analyte.

It is noted that at least one of the first and second analyte bindingagents should bind to the analyte and not bind to any of the othercomponents in the sample to be analyzed, referred to herein as ananalyte-selective binding agent. In one embodiment, the first analytebinding agent which is immobilized in a test zone is ananalyte-selective binding agent and the second analyte binding agentwhich is labeled with a detectable marker is capable of bindingnon-selectively to the analyte. In another embodiment, the first analytebinding agent which is immobilized in a test zone is capable of bindingnon-selectively to the analyte and the second analyte binding agentwhich is labeled with a detectable marker is an analyte-selectivebinding agent. In yet another embodiment, both the first and secondanalyte binding agents are analyte-selective binding agents.

Examples of analyte-selective binding agents include antibodies(monoclonal, polyclonal, and fragments thereof) which have a narrowbinding affinity to only a particular type of biomolecule, such as aprotein or receptor. The detectable marker attached to the secondanalyte binding agent may comprise a wide variety of materials, so longas the marker can be detected. Examples of detectable markers include,but are not limited to particles, luminescent labels; colorimetriclabels, fluorescent labels; chemical labels; enzymes; radioactivelabels; or radio frequency labels; metal colloids; and chemiluminescentlabels. Examples of common detection methodologies include, but are notlimited to optical methods, such as measuring light scattering, simplereflectance, luminometer or photomultiplier tube; radioactivity(measured with a Geiger counter, etc.); electrical conductivity ordielectric (capacitance); electrochemical detection of releasedelectroactive agents, such as indium, bismuth, gallium or telluriumions, as described by Hayes et al. (Analytical Chem. 66:1860-1865(1994)) or ferrocyanide as suggested by Roberts and Durst (AnalyticalChem. 67:482-491 (1995)) wherein ferrocyanide encapsulated within aliposome is released by addition of a drop of detergent at the detectionzone with subsequent electrochemical detection of the releasedferrocyanide. Other conventional methods may also be used, asappropriate.

It may be desired to assay two or more different analytes using the sametest strip. In such instances, it may be desirable to employ differentdetectable markers on the same test strip where each detectable markerdetects a different analyte. For example, different detectable markersmay be attached to different analyte-selective binding agents. Thedifferent detectable markers may be different fluorescent agents whichfluoresce at different wavelengths.

When detecting two or more different analytes using the same test strip,separate test zones may optionally be formed on the test strip for eachanalyte to be detected. The same detectable marker may be used for allof the analytes. Alternatively, different detectable markers, asdescribed above, may be used for the different analytes in order toprevent one test zone being confused with another.

In a preferable embodiment, the detectable marker is a particle.Examples of particles that may be used include, but are not limited to,colloidal gold particles; colloidal sulphur particles; colloidalselenium particles; colloidal barium sulfate particles; colloidal ironsulfate particles; metal iodate particles; silver halide particles;silica particles; colloidal metal (hydrous) oxide particles; colloidalmetal sulfide particles; colloidal lead selenide particles; colloidalcadmium selenide particles; colloidal metal phosphate particles;colloidal metal ferrite particles; any of the above-mentioned colloidalparticles coated with organic or inorganic layers; protein or peptidemolecules; liposomes; or organic polymer latex particles, such aspolystyrene latex beads.

A preferred class of particles is colloidal gold particles. Colloidalgold particles may be made by any conventional method, such as themethods outlined in G. Frens, 1973 Nature Physical Science, 241:20(1973). Alternative methods may be described in U.S. Pat. Nos.5,578,577, 5,141,850; 4,775,636; 4,853,335; 4,859,612; 5,079,172;5,202,267; 5,514,602; 5,616,467; 5,681,775.

The selection of particle size may influence such factors as stabilityof bulk sol reagent and its conjugates, efficiency and completeness ofrelease of particles from conjugate pad, speed and completeness of thereaction. Also, particle surface area may influence steric hindrancebetween bound moieties. Particle size may also be selected based on theporosity of the membrane strip. The particles are preferablysufficiently small to diffuse along the membrane by capillary action ofthe conjugate buffer.

Particles may be labeled to facilitate detection. Examples of labelsinclude, but are not limited to, luminescent labels; colorimetriclabels, such as dyes; fluorescent labels; or chemical labels, such aselectroactive agents (e.g., ferrocyanide); enzymes; radioactive labels;or radio frequency labels.

The number of particles present in the test strip may vary, depending onthe size and composition of the particles, the composition of the teststrip and membrane strip, and the level of sensitivity of the assay. Thenumber of particles typically ranges between about 1×10⁹ and about1×10¹³ particles, although fewer than about 1×10⁹ particles may be used.In a preferred embodiment, the number of particles is about 1×10¹¹particles.

3. Control Test Zones

As illustrated in FIG. 1, a plurality of test zones 110, 112, 114 may beincluded on the test strip. Each test zone is located such that anautomatic or semi-automatic analytical instrument, or a human reader,may determine certain results of the lateral flow assay.

As discussed previously, immobilized in at least one of the test zonesis a first analyte binding agent which is capable of binding to ananalyte in the sample which the test strip is designed to detect. Insome embodiments, it may be desirable for some of the other test zonesto serve as one or more control zones where one or more control bindingagents have been immobilized. Control agents capable of binding to thecontrol binding agent may be positioned on the test strip at variouslocations or added to the test strip when the assay is being performed.The control agents are preferably labeled with a detectable marker, suchas the detectable markers described above, to facilitate detection ofthe control agent binding to the control binding agent immobilized in acontrol zone.

The control agents and control binding agents may be used in combinationto perform a variety of control functions. For example, the controlbinding pairs may be used to confirm whether the sample and buffer havediff-used properly within the test strip. The control binding pairs arealso employable as internal standards and allow analyte measurementresults to be compared between different test strips. This can be usedto correct for strip-to-strip variability. Such correction would beimpractical with external controls that are based, for example, on astatistical sampling of strips. Additionally, lot-to-lot-and run-to-runvariations between different test strips may be minimized by the use ofcontrol binding pairs. Furthermore, the effects of non-specific binding,as discussed further below, may be reduced. All of these corrections aredifficult to accomplish using external, off-strip controls.

A wide variety of agents are known in the art which may be used as amember of the control binding pair. For example, at least one member ofthe control binding pair may be a naturally occurring or engineeredprotein. The control binding pair may also be a receptor-ligand pair.Additionally, at least one member of the control binding pair may be anantigen, another organic molecule, or a hapten conjugated to a proteinnon-specific for the analyte of interest. Descriptions of other suitablemembers of control binding pairs may be found in U.S. Pat. No.5,096,837, and include IgG, other immunoglobulins, bovine serum albumin(BSA), other albumins, casein, and globulin.

Desirable characteristics for control agent-control binding agent pairsinclude, but are not limited to stability in bulk, non-specificity foranalyte of interest, reproducibility and predictability of performancein test, molecular size, and avidity of binding for each other.

In a preferred embodiment, members of the control binding pair do notbind to anything that might be present in the test strip, e.g., from thesample. In one embodiment, the control binding agent comprises rabbitanti-dinitrophenol (anti-DNP) antibody and the control agent includes adinitrophenol conjugated to BSA (bovine serum albumin).

In one preferred embodiment, the second analyte binding agent and thecontrol agent are each separately bound to different particles.

In another preferred embodiment, both the second analyte binding agentwhich diffuses along the test strip and the control agent are attachedto a single species of particle. Attachment may be by non-specificabsorption or by traditional conjugate chemistries. Alternatively, anon-covalent binding system, such as biotin-avidin, or even an antibodyspecific for the second analyte binding agent may be used to attach theanalyte binding agent to the particle. Bifunctional and multifunctionalreagents may also be used to couple to the second analyte binding agentand the control agent to the particle.

The number of second analyte binding agents and control agents attachedto each particle can be varied, depending on what is appropriate for aparticular assay. For example, two copies of the second analyte bindingagent and one copy of the control agent may be attached to eachparticle. Alternatively, one copy of the second analyte binding agentand two copies of the control agent may be attached to each particle.Other variations on the ratios between second analyte binding agent:control agent: particle can be used depending on the particular assay inwhich they are to be employed, these variations being intended to fallwithin the scope of the present invention.

When the test strip includes more than one control zone, the controlzones may be used to create a calibration curve against which a widevariety of analyte measurement results may be compared. The controlzones may also be used to troubleshoot whether the test strip operatedappropriately.

In one embodiment, the test strip has at least two control zones thathave about the same concentration of control binding agent. It is notedthat it is typically easier and more economical to deliver the sameamount of control binding agent to different control zones.

Alternatively, the concentration of control agent in one of the controlzones may be greater than the concentration of control agent in anotherof the control zones. In this instance, the amount of control bindingpairs will be higher in the control zone with the higher concentrationof control binding agent than in the other control zone.

Incorporating more than one control zone on a test strip can be used toprovide the test strip with a wider dynamic range than conventionallateral flow assays. In preferred embodiments, test strips with 2, 3 ormore control zones are used with a relative scale methodology thatpermits mapping of amounts of control binding pairs detected onto thesame scale on which amounts of analyte detected are reported.

Incorporating more than one control zone on a test strip can also beused to evaluate the performance of the test strip. For example, anapparatus for evaluating an analyte in a sample can determine a ratiobetween the measured amounts of control binding agent in at least twocontrol measurement zones, and detect whether an error has occurred in atest strip based on whether the determined ratio falls outside ofpredetermined acceptable maximum and minimum ratio ranges for that teststrip. If an error is not deemed to have occurred, the apparatus mayproceed to evaluate the amount of analyte in the sample. If an error isdeemed to possibly have occurred, the instrument may notify aninstrument operator of the potential error.

When the concentrations of control binding agent in the first and secondcontrol zones are about the same, the predetermined acceptable maximumand minimum ratio may both be near about 1. When the concentrations ofcontrol binding agent in the first and second control zones aredifferent (e.g., the first control zone has three times as much controlbinding agent as the second control zone), the predetermined acceptablemaximum and minimum ratio may both be near about 3.

An apparatus used to measure the test strips may include executablelogic that determines whether the concentrations of control bindingagent in the first and second control zones are within a predeterminedacceptable maximum and minimum ratio.

An apparatus used to measure the test strips may also include executablelogic that evaluates the amount of analyte in the sample based on acombination of the measured amount of first analyte bound in the analytemeasurement zone and the measured amount of control binding agent boundin the first control measurement zone.

The apparatus may include executable logic that evaluates the amount ofanalyte in the sample based on a combination of the measured amount offirst analyte bound in the analyte measurement zone and the measuredamount of control binding agent bound in the first and second controlmeasurement zones.

The amount of control binding pairs in a given control zone may bemapped onto the same measurement scale on which the amount of analyte isreported, a calibration curve may be drawn through the values of thebinding pairs in the high and low control zones.

When more than two control zones are present, a curve may be generatedthat reflects any nonlinearities present in the assay between the amountof analyte detected and the measurement against which the amount mightbe mapped. While such nonlinearities might otherwise affect assays thatassume a relatively linear relationship, they can be corrected for usingmultiple control zones. 2, 3 or more control zones may be used.

In another embodiment, a single control zone may comprise more than onetype of control agent. This may be of use in embodiments where there aremore than one population of analyte binding agents and analytenon-specific agents coupled to a detection agent. For example, when itis desired to assay two or more analytes of interest on the same assaystrip, two populations of analyte binding agents and analytenon-specific agents coupled to a detection agent may be prepared.Different detection agents may be used for each population, allowing adistinction to be drawn between results for the two different analytesof interest. In such circumstances, it may be desirable to use controlzones comprising different control agents or control binding pairs.

The control zones may be located in a variety of locations within thegroup of test zones. It is noted that the test zones may be placed onvarious locations on the test strip, depending on the flow design of thetest strip consistent with the present invention. In a preferredembodiment, the control zones are adjacent the test zones used to detectanalytes in the sample. In a particularly preferred embodiment, at leastone control zone is positioned proximal to a test zone used to detect ananalyte in a sample and at least one control zone is positioned distalto that test zone.

By positioning the analyte test zone between two control zones, thecontrol zones can be effectively used to confirm several operations ofthe test strip. For example, the control zones can confirm that bufferwas added and that sufficient buffer was added so that the buffercompletely traversed the analyte test zone in both directions.Development of the analyte test zone confirms that the sample was added.By measuring a relationship between the control zones, it is alsopossible to confirm that sufficient sample was added and that the stripflowed properly.

Assays are performed using a test strip which includes one or morecontrol regions as part of the test regions in the same manner asdescribed in regard to FIGS. 2A-2H and 3A-3H. It is noted that eitherthe test strip or the buffer may include the control agent which bindsto the control binding agent immobilized, for example, in test zones 110and 114 of FIGS. 2A-2H and test zones 210 and 214 of FIGS. 3A-3H. Whenthe buffer is added, the control agent diffuses with the buffer andbinds to the control binding agent immobilized in the control zones.When the sample is added, the sample serves to wash any unbound controlagent away from the control zone.

Amounts of control agents immobilized in the control zones are detectedalong with the detection of amounts of second analyte binding agentimmobilized in the test zones. As noted above, it is preferred for thecontrol agents and the second analyte binding agent to be labeled with adetectable marker which facilitates their detection. The amount ofdetectable marker in each test zone can be readily determined by avariety of techniques known in the art, depending on the type ofdetectable markers being employed. Common examples of detectiontechniques include optical methods (light scattering, simplereflectance, luminometer or photomultiplier tube); radioactivity;electrical conductivity; dielectric capacitance; electrochemicaldetection of released electroactive agents; as has been noted above.

Once the amount of detectable markers has been measured in each testzone, these measurements may be used to detect and preferably quantifythe amount of analyte present, preferably by also calibrating the teststrip using the amounts of detectable markers in the control zones. Forexample, when one or more control zones are employed, the amount ofcontrol agent immobilized in one or more of the control zones may beused to quantify the amount of first analyte binding agent relative toone or more of the control zones. These relative intensity measurementsmay then be used to more accurately determine the number of copies ofanalyte present in the measurement volume.

One feature of using multiple control zones is the ability to create arelative scale for analyte measurements. Once the amounts of detectablemarkers have been quantified, these amounts may then be mapped ontoanother measurement scale. For example, while the results from measuringthe analyte may be measured based on an absolute measurement of theanalyte, the results reported may be more meaningful in other units,such as an intensity relative to that of a control zone or controlzones, referred to herein as Relative Intensity or RI. Results may alsobe expressed as the number of copies of analyte present in themeasurement volume. The mapping of the amount of analyte detected ontoother measurement scales is a preferable embodiment for reportingresults of the inventive assay.

In addition to reporting the assay results on a continuous scale, eitherdirectly as the amount of analyte detected or indirectly as ameasurement scale onto which the amount of analyte detected has beenmapped, the inventive assays may be used in a “cut-off” style assay. Ifthe detectable marker is detected in an analyte binding zone, the amountof detectable marker detected may be compared against a cut-off value. Acut-off value is the value above which the test may be consideredpositive; that is, the analyte of interest is present in the fluidsample to some degree of statistical confidence. Below the cut-offvalue, the test is generally considered not positive—either the analyteof interest is not present or the inventive lateral flow assay did notdetect its presence. While a cut-off may established based upon adirectly measured value, such as the amount of analyte detected, theresults may be more meaningful if reported on an indirect, or relative,scale.

A cut-off lateral flow assay is more desirable as the measurementseparation between a negative value and a positive value increases. Anegative value is the reported value on the continuous scale in the casewhere the analyte of interest is statistically not present. Conversely,a positive value is the reported value on the continuous scale in thecase where the analyte of interest is statistically present. As thesevalues converge, the likelihood reduces of being able to statisticallytell positives and negatives apart.

Also desirable is a cut-off lateral flow with increased precision at thecut-off. When there is less variation at the chosen cut-off, it is morelikely that a positive can be accurately considered a positive and anegative be accurately considered a negative.

Assay results may be mapped onto either a “relative,” discussed above,or an “absolute” scale. Absolute scales are measured in actual physicalunits, such as number of copies of analyte per milliliter of fluid.Measurement in the absolute scale may be preferable in testing forcertain diseases or conditions, such as tests for cancer markers, suchas for PSA or hormones such as TSH. In such preferable embodiments, theresult may be expressed in units, such as ng/ml. Accordingly, thecontrol zones may have value assigned concentrations of control agent.In an extension of the relative measurement concept, the density ofreflectance (DR) values of a series of standards of known analyteconcentration may be measured and the intensities relative to thecontrols (RI values) calculated as previously described. The RI valuesmay then be plotted against analyte concentration to construct astandard curve in which the RI values are assigned concentration valuesof the analyte of interest. The RI of a sample may then be read on thisvalue assigned standard curve, yielding a result labeled in the desiredunits.

Many circumstances may affect the absolute reactivity of lateral flowassays, including, but not limited to, reagent flow variations,manufacturing-derived variations, operator induced variations,environmentally induced variations and sample effects. With conventionallateral flow assays, any of these variations may act to repress orarguably enhance reactivity of one strip over another, resulting inpossible false negative or false positive results. Not controlling forthese or other variations may result in significant imprecision,non-reproducibility, lack of sensitivity and lack of specificity of thetests.

Lateral flow assays are also subject to a number of interferences whichmight affect the absolute amount of binding of either analyte bindingagent or control agent to the test zones. Influencing factors mayinclude: 1) variability in the release of the first analyte bindingagent or the control agent from a conjugate pad, 2) device to devicevariation in the non-specific binding of the analyte binding populationto the test strip, 3) variability in the movement of the analyte bindingpopulation through or along the test strip during the assay due tovariations in reagent flow rates between when a portion of the strip isdry and when a portion of the strip is wet, variations in the pore sizeof the test strip or membrane strip materials or non-specificaggregation of the analyte binding agent. Variability of absolutemeasurements of binding due to these or other factors may therefore beunacceptably high in conventional lateral flow assays.

The use of control zones on test strips is also described in greaterdetail in application Ser. No. 09/198,118, filed Nov. 23, 1998 andapplication Ser. No. 09/638,668, filed Aug. 14, 2000, which are eachincorporated herein by reference.

Examples 1. Construction of Test Strip

In this example, the construction of a test strip having a design asillustrated in FIGS. 1 and 7 is described. Backed sheets ofnitrocellulose, for example, Millipore STHF or HF 90 nitrocellulose (4.8cm×20 cm) were coated by longitudinally dispensing one antigen test bandand two control bands onto the nitrocellulose using a Biodot Quanti-3000XYZ Dispensing Platform with Biojets operating at a frequency of 180Hz., 20.83 nl/drop and 0.75 μl/cm. The nitrocellulose sheets were thendried for one hour at 37° C. in a forced air incubator. Coatednitrocellulose sheets were stored desiccated at room temperature in foilpouches.

Gelman 8980 glass fiber pads, for use as conjugate pads, were preblockedby dipping in a solution of PBS containing 10 mg/ml BSA, 1% (w/v) TritonX-100, 2.5% (w/v) sucrose, 0.3% (w/v) polyvinyl pyrrolidone K-30 and 2mg/ml rabbit IgG. The preblocked pads were then dried for two hours in aforced air incubator. A solution of control and test conjugates in PBScontaining 10 mg/ml BSA, 1% (w/v) Triton X-100, 2.5% (w/v) sucrose, 0.3%(w/v) polyvinyl pyrrolidone K-30 and 2 mg/ml rabbit IgG waslongitudinally dispensed on the preblocked conjugate pads using a BiodotQuanti-3000 XYZ Dispensing Platform with Biojets operating at afrequency of 120 Hz., 104.17 nl/drop and 2.5 μl/cm. The conjugate padswere coated with conjugate in patterns of from one to four lines per cmwith one pattern coated on each 1.3 cm×20 cm pad. Coated conjugate padswere vacuum dried at 2 Torr for two hours at room temperature.

Cytosep 1662 sheets, for use in preparing sample pads, were preblockedby dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v) Triton X-100,2.5% (w/v) sucrose and 0.3% polyvinyl pyrrolidone K-30. The sheets werethen dried for two hours in a forced air incubator. After drying sheetswere slit to strips 7.5 mm wide using a G&L Precision Die Cutting DrumSlitter.

Cytosep 1662 sheets, for use in preparing conjugate buffer pads, werepreblocked by dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v) TritonX-100, 2.5% (w/v) sucrose, 0.3% polyvinyl pyrrolidone K-30, 2 mg/mlRabbit IgG, 1 mg/ml Goat IgG and 0.33 mg/ml heterophyllic blockingreagent 1 (HBR-1) then drying for two hours in a Forced air incubator.The sheets were then slit to strips 0.75 cm wide using a G&L PrecisionDie Cutting Drum Slitter and further cut to 0.75 cm×1.2 cm pads using aBiodot Guillotine cutter.

Test strips were prepared by affixing one 4.8 cm×20 cm backednitrocellulose sheet, and one 1.3 cm×20 cm coated preblocked conjugatepad onto one adhesive coated 0.010″ thick 6 cm×20 cm vinyl backing sheet(G&L Precision Die Cutting). One 0.75 cm×20 cm sample pad was thenaffixed to the nitrocellulose using double sided adhesive. Strips 0.5 cmwide were cut from the assembled sheet with a Kinematics AutomationMatrix 2360 Guillotine Cutter. To assemble the test strip into a testcartridge, illustrated in FIGS. 5A and 5B, the strip was placed in thebottom half of the holder and a 0.6 cm×2.7 cm absorbent pad was placedover the top of the strip. A 0.75 cm×1.2 cm preblocked conjugate bufferpad was then placed over the conjugate pad and aligned with the bottomof the strip and the pins of the top half of the holder aligned with theholes of the bottom half and the holder tightly pressed together.

2. Thyroid Stimulating Hormone (TSH) Assay

Strips used in this example were coated with 3 mg/ml rabbitanti-dinitrophenyl (anti-DNP) in the high control band and 0.8 mg/mlrabbit anti-dinitrophenyl in the low control band and 4 mg/ml affinitygoat anti-TSH in the antigen band on Millipore SRHF nitrocellulose. Theorder of the bands on the strip was low control zone closest to thesample addition pad, high control zone farthest from the sample additionpad (closest to the buffer addition pad) and antigen band (anti-TSH)between the low control zone and the high control zone. Nitrocellulosesheets were coated and strips prepared as in Example 1.

Preblocked conjugate pads were coated with a mixture of 0.2 volumes ofAnti-DNP-32 nm gold conjugate (OD 520 nm approximately 83) and 0.13volumes of monoclonal anti-TSH 32-nm gold conjugate (OD 520 nmapproximately 102) in a total of four volumes of PBS containing 10 mg/mlBSA, 1% (w/v) Triton X-100, 2.5% sucrose and 0.3% (w/v) polyvinylpyrrolidone K-30. The mixture was dispensed onto preblocked conjugatepads as described in Example 1.

The assay was carried out by placing the cassette on the lab bench andthen adding 40 μl of release buffer (5.5×PBS, 10 mg/ml BSA, 0.025%casein, 0.325% Tween20, 2 mM EDTA, 0.1% sodium azide) containing 160pg/ml BSA-DNP to the sample addition port of the cassette. The cassettewas immediately placed in a ReLIA™ machine set up to run and read theReLIA™ assay for the detection of TSH. At the prompts, sample number andassay time were entered, triggering the sample addition clock. After atime sufficient for prewetting of the strip to a point distal to the lowcontrol zone (56 seconds, machine time constant of 40), the machineprompted the user to add 150 μl of the samples shown in FIG. 8 to theconjugate buffer port of the cassette. Strip temperature was set to 30°C. and the strips were read after 20 minutes. Relative intensity valuesof the samples were generated by calculating the ratio of the density ofreflectance of the antigen band to the density of reflectance of thehigh control band.

Table 1 below provides a definition for the various figure headingsdescribing the test results shown in FIG. 8 as well as in FIG. 10.

TABLE 1 HC(Dr) Raw density of reflectance value for the high controlband LC (Dr) Raw density of reflectance value for the low control bandHC/LC Ratio of the high control band to the low control band. (thisratio is used as a quality control check for each individual strip run)Specimen/HC Ratio is the actual value used by the software to generatethe result. Using this RI ratio helps to normalize strip to stripvariability. μIU/mL Micro international units per milliliter - thequanti- tative level of PSH in the sample calculated from the assaystandard curve

As shown in FIG. 9, a standard curve was calculated from the datasummarized therein relating the RI value given by a TSH standard to theTSH concentration of the standard. This standard curve was then used todetermine the mean TSH concentration of an unknown sample, the standarddeviation on the mean and percent CV, using sixteen strips, each from adifferent coated nitrocellulose sheet, from a single lot of the ReLIA™TSH assay. As shown in FIG. 9, the percent coefficient of variation onthe mean TSH concentration of 8.87 micro International units permilliliter, determined by the ReLIA™ TSH assay, was 5.2% demonstratingthe high reproducibility of the RELIA™ TSH assay.

3. Prostate Specific Antigen (PSA) Assay

Strips used in this example were coated with 3 mg/ml rabbitanti-dinitrophenyl (anti-DNP) in the high control band and 1.0 mg/mlrabbit anti-dinitrophenyl in the low control band and 4 mg/ml affinitygoat anti-PSA in the antigen band on Millipore HF 135 nitrocellulose.The order of the bands on the strip was low control zone closest to thesample addition pad, high control zone farthest from the sample additionpad (closest to the buffer addition pad) and antigen band (anti-PSA)between the low control zone and the high control zone. Nitrocellulosesheets were coated and strips prepared as in Example 1.

Preblocked conjugate pads were coated with a mixture of 0.2 volumes ofAnti-DNP-32 nm gold conjugate (OD 520 nm approximately 83) and 0.14volumes of monoclonal anti-PSA 32-nm gold conjugate (OD 520 nmapproximately 106) in a total of four volumes of PBS containing 10 mg/mlBSA, 1% (w/v) Triton X-100, 2.5% sucrose and 0.3% (w/v) polyvinylpyrrolidone K-30. The mixture was dispensed onto preblocked conjugatepads as described in Example 1.

The assay was carried out by placing the cassette on the lab bench andthen adding 40 μl of release buffer (5.5×PBS, 10 mg/ml BSA, 0.025%casein, 0.325% Tween 20, 2 mM EDTA, 0.1% sodium azide) containing 160μg/ml BSA-DNP to the sample addition port of the cassette. The cassettewas immediately placed in a ReLIA™ machine set up to run and read theReLIA™ assay for the detection of PSA. At the prompts, sample number andassay time were entered, triggering the sample addition clock. After atime sufficient for prewetting of the strip to a point distal to the lowcontrol zone (56 seconds, machine time constant of 40), the machineprompted the user to add 150 μl of the samples shown in FIG. 10 to theconjugate buffer port of the cassette. Strip temperature was set to 30°C. and the strips were read after 15 minutes. Relative intensity valuesof the samples were generated by calculating the ratio of the density ofreflectance of the antigen band to the density of reflectance of thehigh control band.

As shown in FIG. 11, a standard curve was calculated from the datasummarized therein relating the RI value given by a PSA standard to thePSA concentration of the standard. This standard curve was then used todetermine the mean PSA concentration of an unknown sample, the standarddeviation on the mean and the percent CV, using sixteen strips, eachfrom a different coated nitrocellulose sheet, from a single lot of theReLIA™ PSA assay. As shown in FIG. 11, the percent coefficient ofvariation on the mean PSA concentration of 9.00 nanograms permilliliter, determined by the ReLIA™ PSA assay, was 8.9% demonstratingthe high reproducibility of the ReLIA™ PSA assay.

4. ReLIA TSH (Thyroid Stimulating Hormone) Stop Flow Assay, Sample Addedat Top and Bottom

In this example, the construction of the test strip used was as follows.In general, the test strip used has a design as illustrated in FIGS. 1and 7. Backed sheets of nitrocellulose, for example, Millipore STHF orHF 90 nitrocellulose (4.8 cm×20 cm) were coated by longitudinallydispensing one antigen test band and two control bands onto thenitrocellulose using a Biodot Quanti-3000 XYZ Dispensing Platform withBiojets operating at a frequency of 180 Hz., 20.83 nl/drop and 0.75μl/cm. The nitrocellulose sheets were then dried overnight at 37° C. ina forced air incubator. Coated nitrocellulose sheets were storeddesiccated at room temperature in foil pouches.

Gelman 8980 glass fiber pads, for use as conjugate pads, were preblockedby dipping in a solution of 10 mM Sodium Borate pH 9.0 containing 0.1%polyethylene glycol (MW 20000) and 5% Trehalose. The preblocked padswere then dried for two hours in a forced air incubator. A solution ofcontrol and test conjugates, in 10 mM Sodium Borate pH 9.0 containing0.1% polyethylene glycol (MW 20000) and 5% Trehalose, was longitudinallydispensed on the preblocked conjugate pads using a Biodot Quanti-3000XYZ Dispensing Platform with Biojets operating at a frequency of 120Hz., 104.17 nl/drop and 2.5 μl/cm. The conjugate pads were coated withconjugate in patterns of from one to four lines per cm with one patterncoated on each 1.3 cm×20 cm pad. Coated conjugate pads were vacuum driedat 2 Torr for two and one half hours at room temperature.

Cytosep 1662 sheets, for use in preparing sample pads, were preblockedby dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v) Triton X-100,2.5% (w/v) sucrose and 0.3% polyvinyl pyrrolidone K-30. The sheets werethen dried for two hours in a forced air incubator. After drying sheetswere slit to strips 7.5 mm wide using a G&L Precision Die Cutting DrumSlitter.

Cytosep 1662 sheets, for use in preparing conjugate buffer pads, werepreblocked by dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v) TritonX-100, 2.5% (w/v) sucrose, 0.3% polyvinyl pyrrolidone K-30, 2 mg/mlRabbit IgG, 1 mg/ml Goat IgG and 0.33 mg/ml heterophyllic blockingreagent 1 (HBR-1) then drying for two hours in a Forced air incubator.The sheets were then slit to strips 0.5 cm wide using a G&L PrecisionDie Cutting Drum Slitter and further cut to 0.5 cm×1.2 cm pads using aBiodot Guillotine cutter.

Test strips were prepared by affixing one 4.8 cm×20 cm backednitrocellulose sheet, and one 1.3 cm×20 cm coated preblocked conjugatepad onto one adhesive coated 0.010″ thick 6 cm×20 cm vinyl backing sheet(G&L Precision Die Cutting). One 0.75 cm×20 cm sample pad was thenaffixed to the nitrocellulose using double sided adhesive. Strips 0.5 cmwide were cut from the assembled sheet with a Kinematics AutomationMatrix 2360 Guillotine Cutter. To assemble the test strip into a testcartridge, illustrated in FIGS. 5A and 5B, the strip was placed in thebottom half of the holder and a 0.6 cm×2.7 cm absorbent pad was placedover the top of the strip. A 0.5 cm×1.2 cm preblocked conjugate bufferpad was then placed over the conjugate pad and aligned with the bottomof the strip and the pins of the top half of the holder aligned with theholes of the bottom half and the holder tightly pressed together.

Strips used in this example were coated with 500 μg/ml DinitrophenylBovine Serum Albumin (BSA-DNP) in the high control band, 100 μg/mlBSA-DNP in the low control band and a 4 mg/ml Affinity Goat anti-TSH inthe antigen band. The order of the bands on the strip was low controlzone closest to the conjugate pad, antigen band between the low controlzone and the high control zone and the high control zone farthest fromthe conjugate pad and closest to the absorbent pad. Nitrocellulosesheets were coated and strips prepared as described above.

Conjugate pads preblocked with 2 mM Sodium Borate pH 9.0 containing 5%Trehalose were coated with a mixture of Anti-DNP conjugate [Rabbitanti-DNP (2×)]-30 nm gold and anti-TSH conjugate [Monoclonal anti-TSH(2×)]-30 nm gold. This was accomplished by mixing 0.3 volumes of theanti-DNP stock conjugate solution (OD 520 approximately 100) and 0.7volumes of the anti-TSH conjugate (OD 520 approximately 124) with twovolumes 2 mM Sodium Borate pH 9.0 containing 5% Trehalose and one volume2 mM Sodium Borate pH 9.0. The mixture was dispensed onto preblockedconjugate pads as described above.

The assay was carried out by placing the cassette on the lab bench andthen adding 50 μl of the sample to the sample pad through the proximalport of the sample cassette. The cassette containing the strip wasplaced in a ReLIA™ machine set up to run and read the ReLIA™ assay forthe detection of TSH. At the prompt additional 100 μl of the sample wasadded to the distal sample port of the cassette. Assay temperature wasset at 30° C. and the strips were read after 20 minutes. Relativeintensity (RI) values of the samples were calculated by dividing thedensity of reflectance of the sample (test) band by the density ofreflectance of the high control band. A standard curve for the TSH assaywas calculated from RI values of TSH standards and programmed into theReLIA™ machine using a 4 parameter logistic curve fit.

As shown in FIG. 12, the performance of the ReLIA™ TSH assay when samplewas added to both the top and bottom ports was compared to that whensample was added only to the bottom port. The data demonstrate that theprecision on the high control density of reflectance was higher whensample was added to the top and bottom ports versus the bottom portalone. This translated into precision on the measured level of TSH whichwas at least equivalent to that obtained when sample was added to thebottom port alone and, in most cases, better.

In FIG. 13 the performance of the ReLIA™ TSH assay using quality controlsamples representing normal (approximately 1 μIU/mL), borderlineelevated (approximately 5 μIU/mL) and elevated (approximately 25 μIU/mL)levels of TSH is displayed. The assay was highly reproducible at allthree TSH levels with CV values under 9%.

As shown in FIG. 14, when the assay protocol employing sample additionfrom both the upper and lower ports was used, ReLIA™ values for TSH inpatient samples, calculated by the machine from the standard curve,correlated well with TSH levels measured by the reference method.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the apparatus and methods ofthe present invention without departing from the spirit or scope of theinvention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents. Additionally,the following examples are appended for the purpose of illustrating theclaimed invention, and should not be construed so as to limit the scopeof the claimed invention.

1. (canceled)
 2. A test strip characterized in that: said test strip isa lateral flow test strip adapted to receive a sample for performance ofan assay to detect an analyte in said sample; and said test stripreduces performance variability during the performance of said assay. 3.The test strip of claim 2, wherein said performance variability isreduced by reducing absolute amount of either an analyte binding agentor a control binding agent that binds to region on said test strip. 4.The test strip of claim 2, wherein said performance variability isreduced by reducing variations in liquid flow rates across said teststrip.
 5. The test strip of claim 4, wherein said performancevariability is reduced by equilibrating said liquid flow rate so thatsaid sample, once added, moves across said test strip at a uniform rate.6. The test strip of claim 2, wherein said performance variability isreduced by prewetting said test strip prior to addition of said sample.7. The test strip of claim 3, wherein said region on said test stripcomprises a test zone, a control zone, or both.
 8. The test strip ofclaim 6, wherein said test strip is prewet with buffer, wherein saidbuffer may, optionally, comprises a control agent.
 9. The test strip ofclaim 8, wherein said buffer is added to a region of said test stripcomprising a buffer addition zone.
 10. The test strip of claim 9,wherein said buffer flows across said test zone, control zone, or both,and then independently flows back toward said buffer addition zone. 11.The test strip of claim 10, wherein said buffer flows back toward saidbuffer addition zone independent of any user intervention.
 12. The teststrip of claim 8, wherein said buffer is added to said test strip withina predetermined volume range designed for said test strip.
 13. The teststrip of claim 12, wherein said predetermined volume range is betweenabout 10 μl and about 250 μl.